以Fe(Ⅲ)改性胶原纤维为载体固定过氧化氢酶

将胶原纤维用三价铁改性后作为载体,通过戊二醛的交联作用将过氧化氢酶固定在该载体上.制备的固定化过氧化氢酶蛋白固载量为16.7 mg/g,酶活收率为35％.研究了固定化酶与自由酶的最适pH、最适温度、热稳定性、贮存稳定性及操作稳定性.结果表明:过氧化氢酶经此法固定化后,最适pH及最适温度与自由酶相同,分别为pH 7.0和25℃;但固定化酶的热稳定性显著提高,在75℃保存5 h后,仍能保留30％的活力,而自由酶则完全失活;固定化酶在室温下保存12 d后,酶活力仍保持在88％以上,而自由酶在此条件下则完全失活;此外,固定化过氧化氢酶还表现出了良好的操作稳定性,在室温下连续反应26次后,相对活力为57％.该研究表明胶原纤维可作为固定化过氧化     

以Fe(III) 改性胶原纤维为载体固定过氧化氢酶

将胶原纤维用三价铁改性后作为载体,通过戊二醛的交联作用将过氧化氢酶固定在该载体上。制备的固定化过氧化氢酶蛋白固载量为16.7 mg/g,酶活收率为35％。研究了固定化酶与自由酶的最适pH、最适温度、热稳定性、贮存稳定性及操作稳定性。结果表明：过氧化氢酶经此法固定化后,最适pH及最适温度与自由酶相同,分别为pH 7.0和25 ℃;但固定化酶的热稳定性显著提高,在75 ℃保存5 h后,仍能保留30％的活力,而自由酶则完全失活;固定化酶在室温下保存12 d后,酶活力仍保持在88％以上,而自由酶在此条件下则完全失     

温和气单孢菌YH311硫酸软骨素裂解酶的分离纯化与固定化

通过硫酸铵沉淀、QAESephadex-A50柱层析及Sephadex-G150凝胶过滤等纯化步骤,对源自温和气单孢菌YH311的ChSase进行了分离纯化。结果表明,ChSase经上述纯化步骤后被纯化了55倍,其最终纯度可达95%以上,比活为31.86u/mg。经SDSPAGE及IFE测定可知该酶的分子量约为80kD,等电点为4.3～4.8。将纯化后的ChSase用海藻酸钠或纤维素固定化后,ChSase的热稳定性及贮存稳定性均可得到大幅度的提高：固定化酶用80℃水浴处理120min或于4℃冰箱放置30d后仍可保留50%以上的相对活力;但固定化酶的收率较低,仅为18.56%和18.86%。     

生物酶法制浆半纤维素酶液的防腐技术研究

研究结果表明,在室温（30±5℃）下,乙酸乙酯、苯甲酸钠对放线菌z-6所产半纤维素酶液有防腐作用,酶液保存10d,酶活剩余50％左右;5．0％的氯化钠对青霉菌m8所产半纤维素酶液有明显防癌作用,酶液保存15d,酶活剩余57．7％。     

Aspergillus sp.脂肪酶发酵条件优化及酶学性质的研究

作者为了得到一种热稳定性较好的脂肪酶新酶种,通过研究分离白极端环境的Aspergillus sp．的最佳产酶条件及其所产脂肪酶的酶学性质,得出了该菌产酶的最佳发酵条件为：以1％黄豆饼粉为氮源、0．2％玉米淀粉为碳源,1．5％橄榄油为诱导物,起始pH6．0左右。装量10mL（250mL三角瓶。摇瓶转速180r／min）、发酵时间为96h。在最佳发酵条件下可得最大发酵酶活36U/mL。Aspergillus sp．所产的脂肪酶的酶学性质是：最适pH为9．0,在pH5．0—10．0于20℃下放置24h后,残余酶活仍保持在起始酶活的90％以上;该酶的最适温度为50℃,50℃保温60min后仍保留70％以上的酶活。Aspergillus sp．所产脂肪酶的热稳定性较好。     

高产耐高温脂肪酶生产菌的筛选与鉴定

从小笼包蒸屉垫中筛选得到了两株脂肪酶高产菌株J2和J3,经形态观察以及26S rRNA基因(26S rDNA)序列比对鉴定,两株菌分别属于Aureobasidium属的两个变体。200 r/min、30℃下摇瓶发酵3-5 d后,以对硝基苯酚棕榈酸酯(p-NPP)作为底物,用分光光度法测得J2和J3发酵上清液中的脂肪酶酶活分别为10.61 U/m L和14.43 U/m L。对两株菌所产脂肪酶的耐热特性研究显示,菌株J2产脂肪酶的最适反应温度为50℃,并且酶液在50℃保温5 h无酶活损失;另一株菌J3所产脂肪酶的最适反应温度为60℃,酶液在50℃保温5 h后酶活剩余42.19%,在40℃保温5 h没有酶活损失。这表明J2和J3菌株所产脂肪酶具有较好的热稳定性和较高的最适反应温度。     

发酵性丝孢酵母蛋白酶稳定性的研究

研究了发酵性丝孢酵母所产蛋白酶的最适作用条件及稳定性。结果表明,发酵性丝孢酵母蛋白酶的最适反应温度为55℃,最适反应p H为7.0;有良好的热稳定性,50℃保温30 min,仍保留78%以上的酶活力;贮存稳定性也较好,10℃贮存60 h,仍保留80%左右的酶活力;5 mmol/L的金属离子Mn2+和Ca2+对蛋白酶有激活作用,其中Ca2+激活作用最显著,使酶活提高了0.87倍;1%(w/w)的表面活性剂吐温-100可以促进酶活力,使酶活提高了24.6%,1%(w/w)的SDS对酶活力有抑制作用,将残余酶活降低为原酶液的67.1%;2.5%(w/w)的葡萄糖、半乳糖和海藻糖等能抑制该蛋白酶活力,其中海藻糖抑制作用最显著,使残余蛋白酶活力降低到原酶液的23.6%;甘油可作为酶保护剂保护蛋白酶稳定性,且浓度为7.5%(w/w)的甘油能明显激活酶活力。     

重组人白蛋白干扰素α-2b融合蛋白的稳定性研究

研究重组人白蛋白干扰素α-2b融合蛋白(rHSA-IFNα-2b)的稳定性.以抗病毒活性和纯度为主要检测指标,考察rHSA-IFNα-2b的热稳定性,酸碱稳定性以及冻融耐受性.rHSA-IFNα-2b在37℃、55℃下放置5h后生物学活性无明显降低,60℃水浴lh即可见活性降低了约40%;pH值(分别为2、3、9)改变时,生物学活性未发生明显变化;rHSA-IFNα-2b反复冻融后聚合体逐渐增加,冻融4次后聚合体含量达到17.91%,但生物学活性无明显降低.重组人白蛋白干扰素α-2b融合蛋白对热、酸碱变化稳定,但不宜冻融.     

一种耐热几丁质酶的产生及其稳定性研究

疏绵状嗜热丝孢菌(Thermomyces lanuginosus)SY2在以胶状几丁质为唯一碳源的诱导培养基中产生了胞外几丁质酶。该酶在50℃保温1 h, 酶活稳定; 65℃时半衰期为25 min; 酶液在室温下保存到12周, 残余酶活性为45％左右。该酶有较宽的pH范围, 3.0~9.0之间保持稳定, pH值为2.5时, 仍具有70％的剩余酶活性。Ca2+ 对几丁质酶的活性有显著的激活作用; 高浓度变性剂对酶有抑制作用。结果表明该酶是一种热稳定性高且耐酸碱的新型几丁质酶, 能在酸性和高温环境中发挥作用, 这些特性赋予了T. lanuginosus几丁质酶在几丁质的生物转化及其它生物技术中极大的应用优势。     

口服脊髓灰质炎减毒活疫苗(人二倍体细胞)的稳定性研究

目的评价细胞工厂工艺连续生产的口服脊髓灰质炎减毒活疫苗(人二倍体细胞)的稳定性。方法疫苗在-20℃放置24个月,检测病毒滴度、外观、抗生素残留量、无菌性,及对病毒血清型进行鉴别;2~8℃放置12个月检测疫苗稳定性;室温放置7周、37℃放置7 d检测加速热稳定性并冻融的稳定性。结果该疫苗-20℃可贮存24个月以上,2~8℃有效期可延长至12个月,且冻融不会影响疫苗的稳定性。结论疫苗质量稳定,各项检测结果均符合《中华人民共和国药典》三部(2010版)及企业《口服脊髓灰质炎减毒活疫苗(人二倍体细胞)注册标准》。     

In vitro heat effect on functional and conformational changes of cyclodextrin glucanotransferase from hyperthermophilic archaea.

The in vitro heat effect on protein characteristics of thermostable enzyme was examined using a cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from the hyperthermophilic archaeon Thermococcus sp. B1001 as a model protein. The recombinant form of CGTase was obtained as an inclusion body from Escherichia coli cells harboring a plasmid which carried the B1001 CGTase gene (cgtA). CGTase was solubilized by 6 M urea, refolded, purified to homogeneity, and heat treated at 80 degrees C for 20 min. Enzyme characteristics were examined compared with those of unheated CGTase. Cyclization activity was increased by in vitro heat treatment, while hydrolysis activity was decreased. The heated and unheated CGTases were analyzed for structures by circular dichroism (CD). The near- and far-UV CD spectra indicated that the structure of unheated CGTase with low cyclization activity was different from that of heated CGTase with high activity. Differential scanning calorimetry of unheated CGTase showed two absorption peaks at 87 and 106 degrees C with increasing temperature. After heat treatment, the minor peak at 87 degrees C disappeared, suggesting that heat-dependent structural conversion occurred in CGTase. These results indicate that the thermal environment plays an important role for the protein folding process of thermostable CGTase.     

Circular dichroism of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus

Circular dichroism (CD) spectra of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus exhibit three positive ellipticity bands between 240 and 300 nm (250, 283, and 292 nm), two negative bands at 327 and 480 nm, and a low-intensity positive band at 390 nm. The fractions of helix β-form, and unordered form of the enzyme are 8, 38, and 54%, respectively. The circular dichroic bands at 327 and 480 nm and a part of the positive bands at 292 and 390 nm are associated with enzyme activity. Significant changes in absorption and CD spectra of the enzyme were observed when the temperature of the enzyme preparation was increased to 47°C, coinciding with the sharp decrease in enzyme activity observed at this temperature.     

诺卡氏菌形放线菌β-甘露聚糖酶的化学修饰及活性中心的研究

分别用 PCMB、NEM、N- AI、NBS等对诺卡氏菌形放线菌β- D-甘露聚糖酶进行化学修饰 ,证明蛋白上的巯基、酪氨酸残基及色氨酸残基是维持酶活性的必需基团 .在加入少量底物后 ,β- D-甘露聚糖酶的最大荧光发射峰从天然状态下的 336nm处蓝移至 332 nm,且峰强度有所增大 .这表明其色氨酸残基隐藏在蛋白内部的疏水区域 .通过对该酶圆二色性扫描光谱的分析 ,表明蛋白内部有二硫键的存在 ;通过巯基乙醇化学修饰的研究 ,表明二硫键是影响该酶热稳定性的一个重要因素 .在蛋白的各种二级结构中 ,α-螺旋、β-折叠、β-转角、自由卷曲的比例分别为 1 6.6%、2 5.4%、2 0 .5%和 37.5% .     

水稻谷胱甘肽磷脂氢过氧化物酶的热稳定性研究

谷胱甘肽磷脂氢过氧化物酶是唯一能够直接还原生物膜上脂类过氧化物的过氧化物酶．本文利用圆二色光谱（CD）、内源荧光光谱和差示扫描量热仪（DSC）研究了温度对谷胱甘肽磷脂氢过氧化物酶（OsPHGPx）活性及其构象变化的影响．在温度为 20－27.5℃ 期间,随着温度的逐渐升高,OsPHGPx 的活性逐渐上升,到 27.5℃ 时达到最大值;在 27.5－45℃ 时,随着温度逐渐升高,其活性迅速下降;当温度超过45℃时,其活性完全尚失. 在20－40℃,CD 光谱、内源荧光光谱和 DSC 均没有发生明显变化,暗示OsPHGPx 的结构基本保持完整;在 40－55℃,CD 光谱显示该酶二级结构发生去折叠;内源荧光光谱的变化暗示该酶三级结构发生去折叠．其中在40－45℃,DSC显示该酶可能存在两个去折叠中间体．当温度超过 55℃ 时,整个酶的构象不再发生变化,呈现去折叠状态．     

Comparative Study and Mutational Analysis of Distinctive Structural Elements of Hyperthermophilic Enzymes

Comparison of the three-dimensional structure of hyperthermophilic and mesophilic β-glycosidases shows differences in secondary structure composition. The enzymes from hyperthermophilic archaea have a significantly larger number of β-strands arranged in supernumerary β-sheets compared to mesophilic enzymes from bacteria and other organisms. Amino acid replacements designed to alter the structure of the supernumerary β-strands were introduced by site directed mutagenesis into the sequence encoding the β-glycosidase from Sulfolobus solfataricus. Most of the replacements caused almost complete loss of activity but some yielded enzyme variants whose activities were affected specifically at higher temperatures. Far-UV CD spectra recorded as a function of temperature for both wild type β-glycosidase and mutant V349G, one of the mutants with reduced activity at higher temperatures, were similar, showing that the protein structure of the mutant was stable at the highest temperatures assayed. The properties of mutant V349G show a difference between thermostability (stability of the protein structure at high temperatures) and thermophilicity (optimal activity at high temperatures).     

Improved thermostability of bacillus circulans cyclodextrin glycosyltransferase by the introduction of a salt bridge

Cyclodextrin glycosyltransferase (CGTase) catalyzes the formation of cyclodextrins from starch. Among the CGTases with known three-dimensional structure, Thermoanaerobacterium thermosulfurigenes CGTase has the highest thermostability. By replacing amino acid residues in the B-domain of Bacillus circulans CGTase with those from T. thermosulfurigenes CGTase, we identified a B. circulans CGTase mutant (with N188D and K192R mutations), with a strongly increased activity half-life at 60 degrees C. Asp188 and Arg192 form a salt bridge in T. thermosulfurigenes CGTase. Structural analysis of the B. circulans CGTase mutant revealed that this salt bridge is also formed in the mutant. Thus, the activity half-life of this enzyme can be enhanced by rational protein engineering.     

Spectroscopic study on the structure and stability of beef liver arginase

The secondary and tertiary structure of the oligomeric arginase (EC 3.5.3.1) from beef liver was investigated by circular dichroism (CD) and fluorescence measurements. The far-ultraviolet CD spectrum of the enzyme at neutral pH is indicative of high helical content. The intrinsic fluorescence emission of the protein is due to tryptophan, the contribution of tyrosine being small. Upon excitation at 295 nm, the maximum of emission occurs at 330 nm, implying that the tryptophan residues are rather buried in a hydrophobic interior of the protein. Ethylenediaminetetraacetic acid (EDTA), which inactivates the enzyme by removing the functional Mn2+-ion from the enzyme, does not dissociate the enzyme into subunits, nor affect noticeably its secondary and tertiary structure. Inactivation occurs in the acid pH range, being complete at pH below 4. However, acidification up to pH 1.5 produced only limited changes in the far-ultra-violet CD spectrum and intrinsic fluorescence emission properties. The enzyme shows noteworthy thermal stability, as shown by measuring the residual activity after heating and by evaluating the temperature dependence of the CD signal at 220 nm and the intensity of emission fluorescence. A temperature of half inactivation (Tm) of 77 degrees was determined upon heating the enzyme at pH 7.5 in the presence of Mn2+-ions for 10 min; in the presence of EDTA, Tm is shifted to 55 degrees. Taken together, these observations indicate that the structural stability of beef liver arginase arises from a clustering of hydrophobic amino acids and from Mn2+-ion binding.     

Characterization of an Archaeal Cyclodextrin Glucanotransferase with a Novel C-Terminal Domain

A gene encoding a cyclodextrin glucanotransferase (CGTase) from Thermococcus kodakaraensis KOD1 (CGT(Tk)) was identified and characterized. The gene (cgt(Tk)) encoded a protein of 713 amino acid residues harboring the four conserved regions found in all members of the alpha-amylase family. However, the C-terminal domain corresponding to domain E of previously known CGTases displayed a completely distinct primary structure. In order to elucidate the catalytic function of the gene product, the recombinant enzyme was purified by anion-exchange chromatography, and its enzymatic properties were investigated. The enzyme displayed significant starch-degrading activity (750 U/mg of protein) with an optimal temperature and pH of 80 degrees C and 5.5 to 6.0, respectively. The presence of Ca(2+) enhanced the enzyme activity and elevated the optimum temperature to 85 to 90 degrees C. With the addition of Ca(2+), the enzyme showed extreme thermostability, with almost no loss of enzymatic activity after 80 min at 85 degrees C, and a half-life of 20 min at 100 degrees C. CGT(Tk) could hydrolyze soluble starch and glycogen but failed to hydrolyze pullulan. Most importantly, although CGT(Tk) harbored a unique C-terminal domain, we found that the protein also exhibited significant CGTase activity, with beta-cyclodextrin as the main product. In order to identify the involvement, if any, of the C-terminal region in the CGTase activity, we analyzed a truncated protein (CGT(Tk)DeltaC) with 23 C-terminal amino acid residues deleted. CGT(Tk)DeltaC displayed similar properties in terms of starch-binding activity, substrate specificity, and thermostability, but unexpectedly showed higher starch-degrading activity than the parental CGT(Tk). In contrast, the cyclization activity of CGT(Tk)DeltaC was abolished. The results indicate that the presence of the structurally novel C-terminal domain is essential for CGT(Tk) to properly catalyze the cyclization reaction.     

Spectral differences between rhodanese catalytic intermediates unrelated to enzyme conformation

Circular dichroism (CD) spectra and UV absorption spectra of two obligatory intermediates in rhodanese catalysis were compared. A broad CD band between 250 and 287 nm increased in a manner stoichiometrically related to the content of enzyme-bound persulfide. Titration of a sample of sulfur-substituted rhodanese (ES) with either cyanide or sulfite gave a stoichiometry that is consistent with one persulfide/molecule of rhodanese (Mr = 33,000). This result agrees with that determined by x-ray crystallography and a method based on quenching of intrinsic fluorescence. Cyanolysis of the persulfide in ES is accompanied by a decrease of UV absorption in the region between 250 and 300 nm. Cyanide titrations followed by the change in absorbance at 263, 272, and 292 nm gave the expected stoichiometry. The magnitude of the difference between the far UV-CD spectra of E and ES found here is smaller than reported previously. This variability suggests that the differences in the secondary structure of these intermediates may not be obligatorily related to the cyanolysis of the persulfide. This view is compatible with recent evidence which suggested that E and ES may be made different by structural relaxation events that occur outside of the catalytic cycle. Furthermore, the methods developed here will be useful in studies on the stability of the catalytic persulfide that has been suggested to be central in the mechanism of several enzymes important in sulfur metabolism.     

Purification and some properties of a thermostable DNA polymerase from a Thermotoga species

A stable DNA polymerase (EC 2.7.7.7) has been purified from the extremely thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1 by a five-step purification procedure. First, the crude extract was treated with polyethylenimine to precipitate nucleic acids. The endonuclease activity coprecipitated. DEAE-Sepharose, CM-Sephrarose, and hydroxylapatite column chromatography were used to purify the preparation. As a final step on a small scale, preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was used. The purified DNA polymerase exhibited a molecular weight of 85,000, as determined by both SDS-polyacrylamide gel electrophoresis and size-exclusion chromatography. Its pH optimum was in the range pH 7.5-8. When assayed over the temperature range 30-80 degrees C, the maximum activity in a 30-min assay was at 80 degrees C. The enzyme was moderately thermostable and exhibited half-lives of 3 min at 95 degrees C and 60 min at 50 degrees C in the absence of substrate. Several additives such as Triton X-100 enhanced thermostability. During storage at 4 degrees C and -70 degrees C, the stability of the enzyme was improved by the addition of gelatin.